GSM8047578: Hippocampus, post-onset 1, snRNAseq; Mus musculus; RNA-Seq
snRNA-seq sample collection was performed on fresh tissue over two batches: batch 1 (pre-onset 1 and control 1) and batch 2 (pre-onset 2&3 and control 2&3)At postnatal day 50 (n = 3 each), mice were euthanized and their brains were removed (Fig 1A-B). Bilateral hippocampi were immediately dissected and placed in a Dounce homogenizer with 1 mL lysis buffer: EZ Prep Lysis Buffer from Nuclei EZ Prep (Sigma #NUC101), 1mM DTT, 27U/mL Protector RNase inhibitor (Sigma #3335399001). Hippocampi were homogenized as follows: 25X with pestle A, 25X with pestle B, wait 2.5 minutes, 15X with pestle B, wait 2.5 minutes, 15X with pestle B. Samples were filtered through 30 um MACS strainers (Myltenyi, 130-098-458) and an additional 1 mL lysis buffer was added. Samples were centrifuged (500 rcf, 5 minutes, 4°C) and supernatant was discarded. Samples were resuspended in 750 uL wash buffer: 5mM MgCl­2, 10mM Tris buffer pH 8.0, 25 mM KCl, 1mM DTT, 27U/mL Protector RNase inhibitor, 1% bovine serum albumin. Samples were filtered through 30 um MACS strainers and centrifuged (500 rcf, 5 minutes, 4°C). Supernatant was discarded and pellets were resuspended in 1 mL wash buffer. Nuclei were stained with propidium iodide at a final concentration of 0.4ug/mL. Sorting was performed at the University of Michigan Flow Cytometry Core on a MoFlo Astrios Cell Sorter (Beckman Coulter). Sorted nuclei were collected in pre-washed tubes containing 100 uL wash buffer. Library preparation and sequencing were performed by the University of Michigan Advanced Genomics Core using the 10X Genomics single-nucleus 3' gene expression platform targeting 10,000 nuclei per sample and 60,000 reads per nucleus. Paired-end sequencing was performed on the NovaSeq6000.